The ketone body β-hydroxybutyrate (BHB) is an endogenous factor protecting against stroke and neurodegenerative diseases, but its mode of action is unclear. Here we show in a stroke model that the hydroxy-carboxylic acid receptor 2 (HCA2, GPR109A) is required for the neuroprotective effect of BHB and a ketogenic diet, as this effect is lost in Hca2−/− mice. We further demonstrate that nicotinic acid, a clinically used HCA2 agonist, reduces infarct size via a HCA2-mediated mechanism, and that noninflammatory Ly-6CLo monocytes and/or macrophages infiltrating the ischemic brain also express HCA2. Using cell ablation and chimeric mice, we demonstrate that HCA2 on monocytes and/or macrophages is required for the protective effect of nicotinic acid. The activation of HCA2 induces a neuroprotective phenotype of monocytes and/or macrophages that depends on PGD2 production by COX1 and the haematopoietic PGD2 synthase. Our data suggest that HCA2 activation by dietary or pharmacological means instructs Ly-6CLo monocytes and/or macrophages to deliver a neuroprotective signal to the brain.
Glucose and the ketone body β-hydroxybutyrate (BHB) are the brain’s energy substrates. An energy deficit plays a causal role in ischemic stroke and has also been involved in other neurodegenerative diseases, such as Parkinson’s disease (PD), Alzheimer’s disease (AD) and amyotrophic lateral sclerosis (ALS)1,2. Therefore, ample supply of glucose and BHB would be expected to ameliorate these diseases. However, the role of glucose and BHB in neurological disorders goes beyond their function as energy substrates. Hyperglycemia aggravates rather than improves ischemic stroke and AD, but the basic mechanisms underlying this paradox are still unclear3. BHB does improve various neurological diseases, though not necessarily by supplying extra energy. Over 90 years of clinical experience have shown that ketogenic diet has anticonvulsive efficacy. In clinical practice, ketogenic diet is now established in the treatment of pharmacoresistant childhood epilepsy. In addition, ketogenic diet and BHB exert a protective function in animal models of stroke, PD, AD and ALS4,5,6,7,8. So far small clinical trials suggest that it is also effective in neurodegenerative diseases9. Currently, larger trials are underway to test ketogenic diet in neurodegenerative diseases (clinicaltrials.gov accession codes NCT01035710, NCT01016522 and NCT01364545). Several mechanisms have been described that explain the antiepileptic efficacy of a ketogenic diet, but the mechanisms underlying its neuroprotective activity have not yet been elucidated4,8,10.
It has long been known that BHB produced by the liver from free fatty acids under fasting conditions reduces in a negative feedback loop the release of fatty acids from adipose tissue11. This important homeostatic function is believed to be mediated by the hydroxy-carboxylic acid receptor 2 (HCA2, GPR109A), a Gi protein-coupled receptor that is activated by BHB on adipocytes12. Interestingly, HCA2 is also stimulated by nicotinic acid and related drugs that are in clinical use to lower plasma lipids and protect against atherosclerotic disorders13,14. HCA2 is not confined to adipocytes but is also present on neutrophils and tissue macrophages14. In addition, expression in the brain has been reported15. Therefore, we investigated the function of HCA2 receptors in the context of neuroprotection induced by ketogenic diet. Our data show that the activation of HCA2 by ketogenic diet, BHB, or nicotinic acid induces a neuroprotective phenotype in bone marrow-derived macrophages that infiltrate the brain and that this results in an improved outcome in a mouse model of stroke.
HCA2 mediates the neuroprotective effect of ketogenic diet
To explore HCA2 function in ketogenic diet-induced neuroprotection, we employed Hca2−/− mice. When we fed wild-type (Hca2+/+) or Hca2−/− mice with a ketogenic diet, body weight did not change (Supplementary Table 1) but plasma concentrations of BHB increased markedly (Fig. 1a). In keeping with the concept that, HCA2 provides a negative feedback on ketone body production by inhibiting fatty acid release11,12, the BHB levels were even higher in Hca2−/− than in wild-type mice (Fig. 1a). Plasma levels of free fatty acids and acetoacetate increased similarly (Supplementary Table 1). We induced ischemic strokes by occluding the distal middle cerebral artery (MCAO). This procedure induces mainly cortical infarcts. Forty-eight hours after MCAO, infarcts were significantly smaller in wild-type mice on a ketogenic diet than in animals on a normal diet (Fig. 1b). Interestingly, the protective effect of the diet was lost in Hca2−/− mice, although they had higher plasma levels of ketone bodies (Fig. 1a,b; Supplementary Table 1). However, there was no significant difference in the infarct size between the genotypes when mice were fed a normal diet.
BHB is the endogenous ligand of HCA2 receptors12. To test whether BHB is involved in reducing the infarct size via HCA2 receptors, we administered BHB by implanting subcutaneous pumps because of its short half-life. This treatment elevated plasma levels of BHB 48 h after MCAO (Fig. 1c). In parallel, BHB treatment improved the stroke-induced neurological deficit as shown by the corner test. In 12 trials, mice tended to turn more often to the contralateral (that is, right) than to the ipsilateral side after MCAO. BHB treatment normalized this preference for the right side in wild-type but not in Hca2−/− mice (Fig. 1d). Furthermore, BHB decreased the infarct volume in wild-type but not in Hca2−/− animals, demonstrating a neuroprotective effect of BHB through HCA2 receptors (Fig. 1e; Supplementary Fig. 1).
Nicotinic acid mimics the effect of ketogenic diet
HCA2 is activated by nicotinic acid, a drug that is used clinically to lower serum lipid concentrations13. In doses equal or lower than those used before in mice to exert clinically relevant effects16,17, nicotinic acid reduced the infarct size and the ischemic disruption of the blood–brain barrier (Fig. 2a,b). Physiological parameters that are known to influence the infarct volume, such as mean arterial blood pressure, blood gases and body temperature were not affected by nicotinic acid treatment (Supplementary Table 2). Laser Doppler flow measurements showed that nicotinic acid did not significantly change cerebral blood flow, although the measurement at a single site may have missed localized effects. Only blood glucose concentrations increased significantly in response to nicotinic acid, which is in line with clinical experience (Supplementary Table 2). However, hyperglycaemia is more likely to diminish the neuroprotective effect of nicotinic acid because elevated glucose concentrations increase the infarct volume in this and in other stroke models3. When we repeated the experiment in wild-type and Hca2−/− mice, nicotinic acid was only effective in wild-type but not in Hca2−/− mice, demonstrating that by activating HCA2 a neuroprotective effect is produced (Fig. 2c). Importantly, nicotinic acid also improved the stroke-induced neurological deficit that was evaluated by three established tests of sensorimotor function18. In the corner test, nicotinic acid treatment normalized the preference for the right side (Fig. 2d). After MCAO, the latency to move one body length was prolonged. Nicotinic acid treatment significantly improved this parameter (Fig. 2e). Finally, in the sticky-tape-removal test, mice needed more time to sense the tape on the contralateral forepaw after MCAO. Nicotinic acid treatment also reduced the time needed to complete this test (Fig. 2f).
In the experiments described so far, nicotinic acid treatment started immediately before MCAO. However, in clinical practice treatment of stroke is often delayed. Therefore, we administered nicotinic acid after onset of MCAO. Although the delayed dosage reduced the efficacy, nicotinic acid still decreased the infarct size significantly when administered up to 4.5 h after MCAO (Fig. 2g).
Cellular localization of HCA2 in the ischemic brain
To localize HCA2 expression in the brain, we used the BAC-transgenic mouse line Hca2mRFP (Gpr109amRFP), in which the Hca2 locus directs the expression of the monomeric red fluorescent protein (mRFP)17. Under normal conditions, mRFP was expressed exclusively by CD11b+ microglia, but not by astrocytes and neurons (Fig. 3a–c). After MCAO, mRFP+ cells accumulated in the periphery of the cortical ischemia (Fig. 3d). They expressed CD11b (87.5±14.6% of mRFP+ cells) and Iba1 (98.7±18.0% of mRFP+ cells), indicating that HCA2 is present on microglia or monocytes/macrophages that infiltrated the ischemic brain. To differentiate these two cell populations, we generated chimeric mice by transplanting wild-type bone marrow to Hca2mRFP mice (WT>Hca2mRFP) and Hca2mRFP bone marrow to wild-type mice (Hca2mRFP>WT). The analysis of WT>Hca2mRFP mice revealed that brain-derived mRFP+ cells that were CD11b+ and correspond to microglia accumulated in the periphery of the infarct (Fig. 3e). In addition, we found evidence that bone marrow-derived monocytes/macrophages express HCA2 in the ischemic brain. In Hca2mRFP>WT animals, numerous bone marrow-derived mRFP+ cells were found in the periphery of the ischemia, whereas scarce mRFP+ cells were present in the nonischemic hemisphere (Fig. 3f). Bone marrow-derived mRFP+ cells expressing CD11b were already detected 24 h after MCAO in the ischemic brain of Hca2mRFP>WT mice, but their number was higher 48 h after MCAO (Figs 3g and 4a–e). A further analysis by flow cytometry demonstrated that most mRFP+ cells in the ischemic hemisphere were CD11b+, CD45+ and Ly-6G− suggesting a monocytic/macrophagic identity (Fig. 4a–g). As reported previously19, Ly-6CHi and Ly-6CLo monocytes/macrophages were present in the ischemic area representing an inflammatory and a resident subset of monocytes/macrophages, respectively20 (Fig. 4f,g). Interestingly, mRFP+ monocytes/macrophages in the ischemic brain were mainly of a Ly-6CLo subtype (Fig. 4f,g; Supplementary Table 3). In contrast, in blood mRFP+ CD45+CD11b+Ly-6G−monocytes spread equally between Ly-6CLo and Ly-6CHi subsets before MCAO (Fig. 5). After MCAO, the number of Ly-6CHi monocytes dropped but Ly-6CLo monocytes remained stable. This suggests that HCA2-expressing Ly-6CLo monocytes preferentially infiltrate the ischemic brain or, more likely in view of previous data19, that Ly-6CHi cells differentiate into this cell type.
Infiltrating monocytes or macrophages mediate the HCA2 effect
To distinguish whether bone marrow-derived monocytes/macrophages or microglia mediate the protective effect of HCA2 in stroke, we generated chimeric mice by bone marrow transplantation (Fig. 6a). As expected, Hca2+/+ mice that received Hca2+/+ bone marrow (Hca2+/+>Hca2+/+) were protected by nicotinic acid (Fig. 6b). Transplantation of Hca2+/+ bone marrow restored the response of Hca2−/− animals (Hca2+/+>Hca2−/−), whereas nicotinic acid lost its activity when Hca2−/− bone marrow was transplanted to Hca2+/+ mice (Hca2−/−>Hca2+/+), which demonstrates that HCA2 in bone marrow-derived cells mediates the protective effect of nicotinic acid (Fig. 6b). The experiment also showed that HCA2 expression in cells that do not derive from bone marrow, such as adipocytes, is not required for the neuroprotective effect of nicotinic acid, implying that the inhibition of free fatty acid release from adipocytes and other metabolic effects of nicotinic acid13 do not contribute to neuroprotection.
To further define the bone marrow-derived cell type that is responsible for the effect of nicotinic acid, we used CD11b-DTR mice that express the human diphtheria toxin receptor under control of the CD11b promoter21. In these mice treatment with diphtheria toxin (DT) ablated blood monocytes/macrophages but not neutrophils19,21. After DT treatment, the number of CD11b+ cells in peripheral blood of CD11b-DTR mice was reduced by 92% in comparison with wild-type animals. As reported previously, ablation of CD11b+ cells by itself did not alter the infarct size (Fig. 6c)19. However, when monocytes/macrophages had been ablated, nicotinic acid no longer reduced the infarct volume (Fig. 6c), demonstrating that the neuroprotective effect of nicotinic acid depends on monocytes/macrophages.
PGD2 is responsible for the neuroprotective effect of HCA2
The activation of HCA2 by nicotinic acid or other agonists is known to stimulate the synthesis of PGD2 (ref. 14). In the skin, PGD2 is responsible for the flushing response induced by nicotinic acid. In the brain, however, PGD2 exerts neuroprotective effects22. Therefore, we investigated whether PGD2 production mediates the neuroprotective effect of HCA2 activation. Haematopoietic PGD2 synthase (HPGDS) and COX1 are responsible for synthesizing PGD2 and its derivatives in macrophages23,24. In accordance with the finding that, HCA2 activation stimulates PGD2 release from macrophages25, plasma concentrations of PGD2 and the renal excretion of tetranor PGDM, a major PGD2 metabolite that reflects the synthesis of PGD2 (ref. 26), increased on nicotinic acid treatment (Fig. 7a,d). Nicotinic acid is able to penetrate the blood–brain barrier27. Twenty-four hours after MCAO, when monocytes/macrophages have infiltrated the ischemic area, we also found elevated PGD2 concentrations in the brain on the activation of HCA2 by nicotinic acid (Fig. 7b). To test the role of PGD2 synthesis in HCA2-mediated neuroprotection, we used Cox1−/− mice and an inhibitor of HPGDS. In our stroke model, the infarct volume in vehicle-treated Cox1−/− mice was similar as in Cox1+/+ animals in line with some but not all previous studies (Fig. 7c)28,29,30. Interestingly, nicotinic acid had no effect on the infarct volume in Cox1−/− mice, whereas Cox1+/+ littermates were protected (Fig. 7c). When we inhibited HPGDS in mice with the small molecule compound HQL79 (ref. 31), renal excretion of the PGD2 metabolite tetranor PGDM was reduced confirming the efficacy of HQL79 as a HPGDS inhibitor (Fig. 7d). In parallel, the protective effect of nicotinic acid was partially reversed by HQL79 (Fig. 7e), suggesting that COX1 and HPGDS mediate the effect of nicotinic acid.
Ketogenic diet and BHB are known to protect against seizures and neurodegeneration. Here we provide evidence that the neuroprotective action of ketogenic diet and of the endogenous ketone body BHB is mediated by HCA2. BHB activates HCA2 with an EC50 of ~\n750 μM (ref. 32), similar to the BHB plasma concentrations of 600–1,000 μM (Fig. 1a,c) that we found to be associated with neuroprotection. In contrast, higher BHB concentrations are required in vitro to protect neurons lacking HCA2 (refs 33, 34, 35). While in our experiments neuroprotection coincided with elevated BHB levels, anti-seizure effects seem to lag behind ketosis36, suggesting that the neuroprotective and the anti-seizure effects are mediated by different mechanisms.
Previous studies showed that HCA2 is activated on adipocytes by BHB and the anti-dyslipidemic drug nicotinic acid12,14. However, the potential of HCA2 to improve CNS disorders was unknown. We found that under normal conditions, microglia express HCA2. Nevertheless, our experiments employing bone marrow transplantation argue against a major role of microglia in HCA2-induced neuroprotection because this cell population is not exchanged by bone marrow transplantation37. Among bone marrow-derived cells, neutrophils and monocytes/macrophages express HCA2 (ref. 14). The activation of HCA2 inhibits adhesion and migration of neutrophils and induces the death of these cells in vitro38,39. Whether modulation of neutrophils contributes to the neuroprotective effects of HCA2 in cerebral ischemia is still unclear. However, the neuroprotective effect of HCA2 depended on monocytes/macrophages (Fig. 6c) that infiltrate the brain in ischemic stroke19. Interestingly, HCA2 was found on a subset of immigrant monocytes/macrophages that was characterized by low expression of Ly-6C, demonstrating that they represent resident monocytes/macrophages20. Recently, this noninflammatory type of monocytes/macrophages has been implicated in protection and repair in stroke and other diseases15,18,40. Our data suggest that HCA2 provides a way to specifically activate Ly-6CLo resident monocytes/macrophages in brain pathology. So far it is unclear whether HCA2 activates Ly-6CLo monocytes/macrophages before brain infiltration in blood or in the ischemic brain. However, the observations that nicotinic acid is able to penetrate into the brain27 and that nicotinic acid treatment leads to elevated PGD2 levels in the brain argue for an effect within the CNS.
The protective effect of HCA2 activation depended on COX1 and HPGDS, the key enzymes that synthesize PGD2 in response to nicotinic acid treatment17,41. While we cannot exclude the possible involvement of COX2 or other arachidonic acid products, our data suggest a new concept by which PGD2 release from monocytes/macrophages mediates the neuroprotective effect of HCA2. Previous work has shown that PGD2 helps to resolve inflammation42. In addition, it has neuroprotective effects22,43. PGD2 has a short half-life in tissue and is spontaneously converted into the cyclopentenone 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), which inhibits the IκB kinase (IKK), the main activator of the transcription factor NF-κB and a key player in ischemic brain damage44,45. In accordance with this, HCA2 activation has been shown to inhibit NF-κB activation in bone marrow-derived macrophages46. 15d-PGJ2 is also an endogenous agonist of PPARγ, a transcription factor with neuroprotective properties47,48. Via this mechanism, nicotinic acid is able to stimulate PPARγ in human monocytes in vitro24. Furthermore, it has been reported that 15d-PGJ2 can stimulate angiogenesis49. Similar mechanisms may underlie the anti-atherogenic effect of HCA2 activation50. In vascular macrophages, HCA2 activation upregulated genes with an anti-inflammatory and anti-atherogenic function50. Collectively, these data suggest that HCA2 provides the pharmacological basis to modulate monocyte/macrophage function and to redirect these cells into a salutary pathway.
Our study demonstrates that HCA2 activation induces a neuroprotective repertoire of resident monocytes/macrophages that can reduce ischemic brain damage. Infiltration of monocytes/macrophages into the diseased brain has also been noted in chronic neurodegenerative disorders, such as AD, PD and ALS, and MS51,52,53, which suggests that the findings we obtained in a model of ischemic stroke may have implications that extend beyond this specific disease. In line with this notion, ketogenic diet and the subsequent production of the endogenous HCA2 agonist BHB are effective in the treatment of several chronic neurodegenerative disorders9. However, compliance is often low because the diet is unpalatable. The discovery of the central role of HCA2 may guide drug development and may ultimately lead to a ‘ketogenic diet in a pill’10. Importantly, the HCA2 receptor is a good target for drug development. The prototypical agonist nicotinic acid has been known to ameliorate ischemic brain damage54,55. More than 50 years ago, before the era of evidence-based medicine, it was used in human stroke patients because of its vasodilatory effects that are apparent from the flushing response in the face56,57,58. However, later studies found little or no vasodilatory effect in cerebral vessels59,60. Thus, its mode of action had been largely unclear. Our data now demonstrate that the neuroprotective activity of nicotinic acid depends on HCA2, very much as that of BHB and ketogenic diet. HCA2 may also mediate the protective effect of dimethyl fumarate and its metabolite monomethyl fumarate in multiple sclersosis38,61. Novel HCA2 agonists showed evidence of a superior potency or of fewer side effects than nicotinic acid during clinical trials testing anti-dyslipidemic effects16,62,63. Thus, synthetic HCA2 agonists provide the pharmacological basis for modulating monocyte/macrophage function and redirecting these important cells into a neuroprotective pathway.
Hca2−/−, Hca2mRFP (Gpr109amRFP), CD11b-DTR, and Cox1−/− mice have been reported previously13,17,21,64. The Hca2−/− mice were backcrossed for more than eight generations on a C57BL/6 background; therefore, we used C57BL/6 mice as wild-type controls. To habituate mice to ketogenic diet (sniff EF R/M ketogenic diet with 80% long-chain fatty acids in dry mass and 8% protein), we mixed it with normal chow (ssniff M-Z, containing 48% carbohydrates, 15% fat and 37% protein) and increased the fraction of ketogenic diet in a stepwise manner (50% for 3 days, 70% for 5 days, 90% for 6 days and 100% for 4 days). All experiments were performed according to the German animal protection law and approved by the local animal welfare authorities (Regierungspräsidium Karlsruhe; Ministerium für Energiewende, Landwirtschaft, Umwelt und ländliche Räume, Kiel, Germany).
In the model, 8- to 15-week-old male mice were subjected to left middle cerebral artery occlusion (MCAO) as described previously65. In brief, the mice were anaesthetized with 15 μl 2.5% tribromoethanol per gram body weight. Panthenol eye ointment was used to prevent eye dryness. A skin incision was made between the ear and the orbit on the left side. The temporal muscle was removed and a burr hole was drilled to expose the stem of the middle cerebral artery (MCA). The MCA was then occluded by microbipolar electrocoagulation (Modell ICC 50, Erbe, Tübingen, Germany). The surgery was done under a microscope (Hund, Wetzlar, Germany), and rectal temperature was maintained at 37 °C during surgery by a heating pad. The skin incision was then closed by suture, and the mice were placed under a heating lamp until they fully recovered. After 24 or 48 h of MCAO, mice were deeply reanaesthetized with tribromoethanol and perfused intracardially with 15 to 20 ml of Ringer’s solution. Brains were carefully removed and coronally cryosectioned (20-μm thick) every 400 μm. Coronal sections were then stained with a silver technique18, and the infarct volume was determined using ImageJ and corrected for brain edema as described previously18,45. Mice were only excluded from analysis if they died before perfusion. The mortality of mice that underwent surgery was 7.6%. To analyse physiological parameters, the femoral artery was cannulated in a separate cohort of animals. Blood samples (100 μl) were withdrawn 10 min before and 10 min after the MCAO. Investigators were blinded to the treatment or genotype of mice or to both in all experiments. Mice were randomized to the treatment groups. If not indicated otherwise, nicotinic acid or vehicle was administered 10 min before MCAO and 4 h, 8 h, 24 h, 28 h and 32 h after MCAO. Because of its short half-life, we administered BHB through subcutaneous Alzet pumps (2001D, releasing 8 μl h−1 BHB, 1 g ml−1, dissolved in normal saline) that were implanted subcutaneously 10 h before MCAO under isoflurane anaesthesia; controls received pumps filled with normal saline. HQL79 was suspended in Methocel (0.2 ml, 0.5%) and administered by gavage 1 h before each nicotinic acid dose. Controls received the vehicle by gavage.
Bone marrow transplantation and ablation of CD11b-positive cells
Bone marrow transplantation was performed as described previously66 with the following modifications. Mice were killed by cervical dislocation, and bone marrow was aseptically collected from femurs and tibias. Unfractionated bone marrow cells were resuspended in 0.25 ml sterile PBS and injected retro-orbitally into 10- to 13-week-old C57BL/6, Hca2−/−, or Hca2mRFP mice that had been lethally irradiated (dose of 10 Gy in 2 divided sessions, 5 Gy each time with a 4-h interval, 10 MV- bremsstrahlung, dose rate of 3 Gy min−1) 1 day before. Six weeks after reconstitution, mice were subjected to MCAO. To ablate CD11b+ cells, we injected CD11b-DTR mice with diphtheria toxin (ip, 25 ng g−1 body weight) 48 h and 24 h before MCAO. Wild-type controls were also treated with diphtheria toxin.
To evaluate sensorimotor function, we used three established tests. The corner and latency-to-move test have been described previously18. In brief, mice were allowed to enter a 30 × 20-cm corner with an angle of 30° before and 48 h after MCAO, and the number of right and left turns on rearing out of 12 trials were counted. For the latency-to-move test, mice were placed at the centre of a plain board. The time to cross one body length (7 cm) was measured before and 48 h after MCAO. In the sticky-tape-removal test, a small circular adhesive tape (HERMA No 2212, 8 mm) was placed onto both forepaws one after another before and 48 h after MCAO, and the time when mice first tried to remove the adhesive tape as well as the total time needed to remove it were determined. In this study, mice were trained before MCAO three times in 3 days, and the last training session was considered as the baseline value.
Flow cytometry was performed as described previously67 with the following modifications. Mice were deeply anaesthetized with tribromoethanol 24 h or 48 h after MCAO and perfused intracardially with Ringer’s solution. Brains were dissected and olfactory bulbs, right hemispheres and cerebella were removed. Left hemispheres were digested in DMEM (Invitrogen) containing collagenase A (1 mg ml−1, Roche) and DNAse (0.1 mg ml−1, Roche) for 30 min at 37°. Then, cells were filtered through a 40-μm nylon cell strainer (BD Biosciences), and the red blood cells were lysed on ice with standard erythrocyte lysis buffer. Myelin and debris were separated from the cell using Percoll gradient (GE Healthcare; 78 and 30%). The cells were collected carefully from the interface of the gradient and washed with 10 ml PBS containing 0.5% BSA. After treatment with purified rat anti-mouse CD16/32 (Fc Block, BD Pharmingen, 1:100) for 10 min on ice, cells were incubated with the antibodies and respective isotype controls for 30 min on ice as follows: PerCP-labelled rat anti-mouse CD45 (BD Pharmingen, 1:100), PE-Cy7-labelled rat anti-mouse Ly-6C (BD Pharmingen, 1:100), APC-labelled rat anti-mouse Cd11b (BD Pharmingen, 1:100) and FITC-labelled rat anti-mouse Ly-6G (BD Pharmingen, 1:100). The cells were then sorted on BD FACS Aria III (BD Bioscience, 100 μm nozzle) with the laser lines 488, 561 and 633 nm.
Twenty-four or 48 hours after MCAO, Hca2mRFP mice were deeply anaesthetized with tribromoethanol and perfused with Ringer’s solution and 4% PFA. Then, 20-μm-thick coronal cryosections were permeabilized with 0.3% Triton X-100 in PBS for 30 min and blocked with 5% BSA. The sections were incubated with rabbit anti-mouse Iba1 (Wako, 1:100), rat anti-mouse CD11b (AbD Serotec, 1:100), mouse anti-NeuN (Chemicon, 1:500) and rabbit anti-GFAP (DAKO, 1.500) overnight at 4 °C. We used the following secondary antibodies to visualize the staining using a confocal microscope (SP5, Leica): Alexa 488-labelled donkey anti-rabbit (Invitrogen, 1:400) and Alexa 488-labelled donkey anti-rat (Invitrogen). The sections were then washed with PBS containing DAPI (Sigma, 1: 5,000) and mounted with Mowiol.
Quantification of IgG extravasation
Cryosections were fixed in 100% acetone for 5 min at −20 °C and incubated for 5 min with 1.5% H2O2 in methanol. After washing, sections were blocked with PBS containing 5% BSA and 0.3% Triton X-100 and incubated with HRP-labelled goat anti-mouse IgG (Santa Cruz) for 1 h. Then, DAB was applied for 5 min according to the manufacturer’s instructions (DAB-Kit, Vector SK-4100). After drying, the sections were mounted with Mowiol. The staining was quantified with ImageJ as integrated density of the ischemic hemisphere relative to the contralateral hemisphere.
Measurement of ketone bodies and PGD2 synthesis.
Total ketone bodies (acetoacetate+BHB), BHB, and free fatty acids in plasma were measured photometrically on an Olympus AU 400 analyzer (Beckman Coulter, Krefeld, Germany) using Autokit Total Ketone Bodies, Autokit 3-HB and NEFA C kit from Wako Chemicals GmbH (Neuss, Germany). The acetoacetate concentration was calculated by subtracting the BHB levels from the total ketone body levels. Plasma and brain concentrations of PGD2 were measured using an ELISA kit (Cayman Chemicals) according to the manufacturer’s instructions. The major PGD2 metabolite tetranor PGDM was measured in urine by ELISA (Cayman Chemicals) at the time of nicotinic acid treatment (baseline level) and four times afterwards at hourly intervals. Tetranor PGDM concentrations were normalized for creatinine levels in urine determined by the creatinine urinary assay kit (Cayman Chemicals) and expressed as percentage of the baseline concentration.
Values in the manuscript are means±s.e.m. For comparison of two groups, we used t-test. For more than two groups one-way analysis of variance (ANOVA) and Newman–Keuls post hoc test or two-way repeated-measures ANOVA followed by Bonferroni post hoc test were employed. On the basis of the s.d. of infarcts that we had determined in previous experiments (25% of means), we chose a sample size of 8 to 10 to detect a 40% reduction of infarct size with a power of 0.8 and an α error of 0.05 when using one-way ANOVA with four groups.
How to cite this article: Rahman, M. et al. The β-hydroxybutyrate receptor HCA2 activates a neuroprotective subset of macrophages. Nat. Commun. 5:3944 doi: 10.1038/ncomms4944 (2014).
We acknowledge the support from the Postdoc program of the Medical Faculty, University of Heidelberg, to S.M. and from the China Scholarship Council to H.C. We thank Anne Strigli and Gudrun Vierke for expert assistance with histology. This study was supported by a grant of the Deutsche Forschungsgemeinschaft to M.S. (SCHW 416/7-1).
Supplementary Figure 1 and Supplementary Tables 1-3